PROJECT SUMMARY Resulting from organ retrieval, cold preservation and a period of warm ischemia during the surgery, ischemia-reperfusion injury (IRI) often leads to primary graft non-function, may predispose to late chronic rejection, and contributes to the shortage of donor organs available for transplantation. At present, there is no specific treatment available to prevent IRI in transplant recipients. Moreover, mechanisms of IR-induced tissue damage, and how this clinically-relevant innate immune response contributes to graft rejection represents one of the most challenging yet understudied problems in clinical liver transplantation. Although IR-hepatocellular damage develops ex-vivo, in the absence of exogenous antigens, and in syngeneic grafts, we and others have documented a crucial role for activated T cells, particularly of CD4 phenotype, in the pathogenesis of liver IRI. T cell exhaustion is a state of T cell dysfunction, characterized by sequential loss of IL-2 production, proliferative capacity, IFN-? production, and finally apoptotic cell death. Previously, we have demonstrated that TIM-3 negative signaling was essential for regaining hepatic homeostasis in ?warm? in-situ, and ?cold? IR-stressed livers. Here, we have identified a new population of TIM-3+CD4+ exhausted T cells, which unlike those described in the literature, may be induced in an exogenous antigen-independent manner, in innate immune milieu of syngeneic mouse livers in response to IR-stress. Hypothesis: Hepatocyte-derived TIM-3 ligands promote the development of TIM-3+CD4+ exhausted T cell phenotype in an exogenous antigen-independent manner, which in turn regulates macrophage pro-inflammatory responses and may promote cytoprotection in IR-stressed syngeneic OLTs. Aim 1: To test mechanisms exacerbating the hepatocellular damage and endogenous TIM-3+ T cell ?exhaustion? phenotype in IR-stressed liver grafts. Hypothesis: TIM-3 ligands (PS, Gal-9, and HMGB1) released from IR-stressed hepatocytes promote generation of TIM-3+CD4+ exhausted T cells without exogenous antigen stimulation. Aim 2: To test the efficacy of TIM-3+ T cell-based ?exhaustion? strategy in promoting liver graft survival. Hypothesis: TIM-3 ligands (PS, Gal-9, and HMGB1) induce TIM-3+CD4+ exhausted T cells, which modulate innate immune activation to prolong the survival of IR-stressed OLTs. By identifying a novel TIM-3+ T cell exhaustion phenotype in antigen-independent milieu of IR-stressed OLTs, this proposal puts forth the advances in molecular delineation of immune exhaustion in a completely new context of innate immune activation. Our findings may have far reaching basic and practical ramifications, as taming IR-triggered very early innate inflammation at the graft site is now considered critical for improving both short and long-term transplantation outcomes.